Gene expression and regulation

An overview

Different types of cells express different sets of genes, thereby exhibiting various shapes and functions.

Central dogma of gene expression

An organism may contain many types of somatic cells, each with distinct shape and function.

However, they all have the same genome.

The genes in a genome do not have any effect on cellular functions until they are "expressed".

Francis Harry Compton Crick

James DeweyWatson

The B form, the helix makes a turn every 3.4 nm, and the distance

Double helix structure of DNA

Hence, there are about 10 pairs per turn. 

Between two neighboring base pairsis 0.34 nm.

In a solution with higher salt concentrations or with alcohol added.

 Z DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. 

I. A form DNA

II. Z form DNA

A-DNA and Z-DNA

The DNA structure may change to an A form, which is still right-handed,

Every 2.3 nm makes a turn and there are 11 base pairs per turn.

Another DNA structure is called the Z form, becauseits bases seem to zigzag.The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure.

The morphology of DNA

Genome

What do you see through the microscope lens below?

This is a real picture of a person's chromosomes - taken from a single cell, stained

-For most organisms, it is the complete DNA sequence. -For RNA viruses, the genome is the complete RNA sequence, since their genetic information is encoded in RNA.

OrganismGenome Size

(Mb)Gene Number

Hepatitis D virus 0.0017 1

Hepatitis B virus 0.0032 4

HIV-1 0.0092 9

Bacteriophage l 0.0485 80Escherichia coli 4.6392 4400

S. cerevisiae (yeast) 12.155 6300

C. elegans (nematode) 97 19000

D. melanogaster (fruit fly) 137 13600

Mus musculus (mouse) 3000 20000-30000

Homo sapiens (human) 3000 20000-30000

“Genome“ is the total genetic information of an organism.

-Chromosomes are compact spools of DNA. -over 3 feet (1 meter) long from end to end if stretch out-enable all this DNA to fit in the nucleus of each cell.-Normally, we have 46 of these packages in each cell;

What are chromosomes and why do we need them?

-Chromatin is the substance which becomes visible chromosomes during cell division.   

The structure of chromatin is dynamically changing, at least in part, depending on the need of transcription.

Chromatin

-Its basic unit is nucleosome, composed of 146 bp DNA and eight histone proteins.

-The 30 nm chromatin fiber is associated with scaffold proteins (notably topoisomerase II) to form loops.

Each loop contains about 75 kb DNA. 

Scaffold proteins are attached to DNA at specific regions called scaffold attachment regions (SARs), which are rich in adenine and thymine.

•G bands are rich in A-T nucleotide pairs

From chromatin to chromosome

The chromatin fiber and associated scaffold proteins coil into a helical structure which may be observed as a chromosome.

•R bands are rich in G-C nucleotide pairs.

-In the metaphase of cell division, the chromatin is condensed into the visible chromosome. 

In the condensed state (linked by H1 and H5)

In the less condensed state (low salt)

Chromatin

-At other times, the chromatin is less condensed, with some regions in a "Beads-On-a-String" conformation.

Overview of DNA Replication

DNA polymerases

Three types of DNA polymerases exist in E. coli: DNA polymerase I, II and III.

A. The DNA polymerase I is used to fill the gap between DNA fragments of the lagging strand. It is also the major enzyme for gap filling during DNA repair.

B. The DNA polymerase II is encoded by the PolB gene, which is involved in the SOS response to DNA damage. C. DNA replication is mainly carried out by the DNA polymerase III.

There are five types of DNA polymerases in mammalian cells: , , , , and . 

DNA polymerase in Mammals

: synthesis of lagging strand. : DNA repair. : synthesis of leading strand. : DNA repair.

The subunit is located in the mitochondria, responsible for the replication of mtDNA. Other subunits are located in the nucleus.

Their major roles :

-Bacteria do not have the end-replication problem, because its DNA is circular.

-to protect chromosomes from fusing with each other

-to solve the end-replication problem. 

Telomerase

In eukaryotes, the chromosome ends are called telomeres which have at least two functions:

Model of Telomerase as an RNA-Reverse Transcriptase ComplexSee Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M., Lundblad, V. and Cech, T. R. Reverse Transcriptase Motifs in the Catalytic Subunit ofTelomerase. Science 276, 561-567 (1997)

Overview of Transcription

Transcription is a process in which one DNA strand is used as template to synthesize a complementary RNA.

(i)Binding of polymerases to the initiation site. 

The entire transcription process

(ii) Unwinding (melting) of the DNA double helix.

Eukaryotic polymerases have to rely on other proteins (transcription factors)

Prokaryotic polymerases can recognize the promoter and bind to it directly,

Unwinding of eukaryotic DNA is carried out by a specific transcription factor.

Prokaryotic polymerases have the helicase activity Eukaryotic polymerases do not.

(iii) Synthesis of RNA based on the sequence of the DNA template strand. 

(iv) Termination of synthesis.  Prokaryotes and eukaryotes use different signals to terminate transcription. 

RNA polymerases use nucleoside triphosphates (NTPs) to construct a RNA strand.

[Note: the "stop" codon in the genetic code is a signal for the end of peptide synthesis, not the end of transcription.

Classes of RNA polymerases

An E. coli RNA polymerase is composed of five subunits: two subunits, and one for each , ', and subunit. 

E. coli:

A. (151 kD) and ' (156 kD) are significantly larger than a (37 kD).

B. Several different forms of subunits have been identified, with molecular weights ranging from 28 kD to 70 kD. The subunit is also known as the factor.

<Q> What’s the function of factor?

There are three classes of eukaryotic RNA polymerases: 

Eukaryotes:

A. I, II and III, each comprising two large subunits and 12-

15 smaller subunits.

D. However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli factor. 

B. The two large subunits are homologous to the E. coli and ' subunits. 

Therefore, in eukaryotes, transcriptional initiation should be mediated by other proteins.

C. Two smaller subunits are similar to the E. coli subunit. 

Both RNA and DNA polymerases can add nucleotides to an existing strand, extending its length. 

RNA polymerases vs. DNA polymerase

RNA polymerases can initiate a new strand but DNA polymerases cannot.

However, there is a major difference between the two classes of enzymes: 

Therefore, during DNA replication, an oligonucleotide (called primer) should first be synthesized by a different enzyme.

RNA processing is to generate a mature mRNA (for protein genes) or a functional tRNA or rRNA from theprimary transcript.

Processing of pre-mRNA involves the following steps:

1. Capping - adding 7-methylguanylate (m7G) to the 5' end. 2. Polyadenylation - adding a poly-A tail to the 3' end. 3. Splicing - removing introns and joining exons.

RNA processing

.

This enzyme changes a codon, CAA, in the middle of theoriginal mRNA to the stop codon UAA, thereby causingearly termination of the protein synthesis.

RNA Editing

-Many prokaryotic mRNAs are polycistronic, namely, an mRNA encodes more than one peptide chain.

Prokaryotes

The 16S rRNA of the ribosome contains a sequence which canpair with the Shine-Dalgamo sequence:

5'—UAAGGAGG (5-10 bases)AUG mRNA 3'--AUUCCUCC........ 16S rRNA

To distinguish them, prokaryotes use a specific sequence located about 5-10 bases upstream of the initiation AUG. 

-The polycistronic mRNA should contain multiple initiating codons.-In the standard genetic code, the codon for both initiating and non-initiating methionine is AUG.

The specific sequence, UAAGGAGG, is known as the Shine-Dalgamo sequence

However, some viral mRNAs are polycistronic or lack 5' cap.

Eukaryotes

5'--ACCAUGG-        mRNA

The mechanism used by eukaryotes to recognize the initiating AUG is not entirely clear.

Eukaryotic ribosome may simply scan from the 5' cap and identify the first AUG as the initiation site.

Nearly all eukaryotic mRNAs are monocistronic (encodes a single peptide).

Marilyn Kozak found that the following sequence may increase the effectiveness as an initiation site

- In bacteria, there are 30-40 tRNAs with different anticodons.

- there are 61 codons coded for amino acids.

- In animal and plant cells, about 50 different tRNAs are found.

- Suppose each codon can pair with only a unique anticodon, - then 61 tRNAs would be needed.

Protein production

Problem